JP2005529579A - Overproduction host for polyketide biosynthesis - Google Patents

Abstract

The present invention provides polyketide overproducing cells that can be used to produce any polyketide and do not require conventional strain improvement. The present invention relates to a method for producing a first polyketide, said method comprising a polyketide synthase (PKS) encoding a PKS producing said first polyketide in a cell optimized to produce a second polyketide. A method comprising the step of expressing a gene is provided. The present invention also includes the steps of producing a first polyketide that is a derivative of the second polyketide, and modifying the PKS gene in the overproducing cell so that the gene expresses PKS producing the first polyketide.

Description

ポリケチド修飾のための遺伝子を含むかまたは含まない上述の遺伝子のうち任意のものは、もしあれば、本発明の過剰産生宿主細胞中で用いられ得る。さらに、本発明の宿主細胞は、所望のＰＫＳの一部および修飾酵素遺伝子クラスターを各々含む複数ベクターとの形質転換によって構築され得る：本明細書中で参考として援用される、米国特許第６，０３３，８８３号を参照のこと。
【００３２】
好ましい様式において、目的のＰＫＳをコードする遺伝子の導入の前または後に、過剰生産株の内在性のＰＫＳ遺伝子は、欠失されるかまたはそうでなければ不活性化される。ある重要な面では、本発明は「過剰産生された」ポリケチドを産生するＰＫＳをコードする遺伝子が欠失されて、そして目的のＰＫＳをコードする遺伝子が容易に導入および発現され得る遺伝的過剰産生宿主細胞を提供する。ある好ましい実施形態では、この遺伝的過剰産生宿主細胞は、エリスロマイシンを２．５ｇ／Ｌを越えるレベルで産生するＳａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ宿主細胞から誘導され、そしてｅｒｙＡ遺伝子の全部もしくは実質的に全部の欠失またはＤＥＢＳ ＰＫＳのモジュール１のケトシンターゼ（ＫＳ）ドメインの変異的不活性化のいずれかによって修飾される。
【００３３】
本発明のこの局面を説明するために、ポリケチドシンターゼＤＥＢＳ上の遺伝ブロックが、６−デオキシエリスロノリドの非産生を生じるＳａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａのエリスロマイシン過剰産生株に導入された。このブロックされた株におけるオレアンドマイシンＰＫＳのｏｌｅＡＩ遺伝子の発現は、１５−ノル−エリスロマイシンＡを産生することが見い出された。このブロックされた変異株の培養物に合成分子が外因性付加することによって、新規な生物学的に活性なエリスロマイシンアナログのＣ−１３誘導体の産生が生じた。この後者の戦略は、前駆体指向型生合成と呼ばれ、ＰＫＳにおける変異の下流に化学的に指向されたプライミング単位または中間体を組込み得る生合成的にブロックされたＰＫＳ変異株を利用する。６−ｄＥＢの多数のＣ−１３誘導体は、Ｓｔｒｅｐｔｏｍｙｃｅｓ ｃｏｅｌｉｃｏｌｏｒにおいて、ＰＫＳに合成的部分鎖の組込むことにより、ＤＥＢＳのモジュール１のケトシンターゼ（ＫＳ１）に変異を導入することによって、産生され得る（本明細書中で参考として援用される、ＰＣＴ公報９９／０３９８６号および同９７／０２３５８号を参照のこと）。しかし、この方法論がＳ．ｅｒｙｔｈｒａｅａの過剰産生株において実行され得る場合、より多くのポリケチドが作製されるというだけではなく、完全にグリコシル化およびヒドロキシル化された化合物が産生されるという点で有利である。
【００３４】
この目的を達成するため、プラスミドｐＫＯＳ４０−５７を送達ベクターとして構築し、ＤＥＢＳ１モジュール１のＫＳ１ドメインに活性部位Ｃｙｓ７２９＞Ａｌａ変異を導入した（前出の、ＰＣＴ公報９９／０３９８６号および同９７／０２３５８号に記載されている）。不活性なＫＳ１ドメインは、開始単位の増殖を防ぎ、そして外因性の合成のジケチドチオールエステルがＰＫＳへ導入されるのを可能にする（本明細書中で参考として援用されるＰＣＴ公報００／０４４７１７を参照のこと）。Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ Ｋ１プロトプラストはｐＫＯＳ４０−５７で形質転換され、そしてＢａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓに対して生物活性を有さない８種の形質転換株が得られた。ｐＫＯＳ４０−５７の組込みが染色体の適切な場所で起こっていることを確認するために、ＰＣＲ分析用に形質転換株の一つからゲノムＤＮＡを抽出した。次いで、二重交差現象を介してのプラスミドの立ち退き（ｅｖｉｃｔｉｏｎ）を可能にする非選択的な成長が行われた。アプラマイシン（ａｐｒａｍｙｃｉｎ）に過敏なコロニーの５分の３が所望のＫＳ１ヌル（ＫＳ１°）変異株にあることが見い出された。Ｋ３９−１４と称される１つのこのようなＫＳ１°ヌル株は、検出可能なエリスロマイシンまたは６−ｄＥＢ様産物をいずれも産生せず、Ｒ２ＹＥ培地中で増殖させた場合に生物活性を示さなかった。このＫ３９−１４株は、ＫＳ１ヌル変異株を補完するｏｌｅＡＩ遺伝子の異種発現のため、および新規エリスロマイシンアナログの前駆体指向型生合成のために使用された。
【００３５】
異種モジュラーＰＫＳ由来のサブユニットは、混成ポリケチドを作製するために機能的に組み立てられ得る。例えば、ＤＥＢＳ３サブユニットおよびＯｌｅＡ３サブユニット（８，８ａ−デオキシオレアンドリドＰＫＳのサブユニット３）の両方は、ピクロマイシンＰｉｋＡＩＩＩおよびＰｉｋＡＩＶサブユニットを完全に補完し得る（本明細書中で参考として援用される、ＰＣＴ公報９９／６１５９９号を参照のこと）。Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ由来の野生型ＤＥＢＳのローディングドメインは、低レベルの１５−ノル−エリスロマイシンｃを形成するためのアセテートを受容することが示されている。ＤＥＢＳがＳｔｒｅｐｔｏｍｙｃｅｓ ｃｏｅｌｉｃｏｌｏｒ中で発現して１５−ノル−６ｄＥＢの（１：５の６ｄＥＢ）を形成する場合に、同様の結果が観察された（本明細書内で参考として援用される、米国特許第５，６７２，４９１号を参照のこと）。Ｏｌｅ ＰＫＳは８，８ａ−デオキシオレアドリドを産生し、これはプライミング単位中の１つの炭素のみだけＤＥＢＳの産物である、６−ｄＥＢとは異なっている。Ｏｌｅ ＰＫＳのサブユニットおよびＤＥＢＳのサブユニットは、有意な構造的相違を含む：これらは、機能的には同様であるにもかかわらず、ＤＥＢＳとＯｌｅ ＰＫＳとの間で４５％しか同一ではない。
【００３６】
発現プラスミドｐＫＯＳ３９−１１０（これは、ｅｒｍＥ^＊プロモーターの制御下で、ｏｌｅＡＩ遺伝子を運搬する）の発現が利用可能であり（本明細書中で参考として援用される、ＰＣＴ公報９９／０６１５９９号を参照のこと）、そしてΦＣ３１結合部位を使用して、染色体内への組込みによって株Ｋ３９−１４内にｏｌｅＡＩ遺伝子を導入するのが好都合であった。Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ Ｋ３９−１４のプラスミドとｐＫＯＳ３９−１１０での形質転換は、株内で生じ、これは副生成物１５−ノル−エリスロマイシンＣを伴って、１５−ノル−エリスロマイシンＡ約５〜１０ｍｇ／Ｌの抗菌活性を示した。マススペクトルおよびＬＣ保持時間は既知の標準と一致した。ｍ／ｚ７０６でのプロトン化分子イオン種は、１５−ノル−エリスロマイシンＣの提案された構造と一致している。この結果は、ＯｌｅＡＩがある程度までＤＥＢＳ１を補完し得ることを示している。また、Ｓ．ｅｒｙｔｈｒａｅａにおけるこの異種ＰＫＳサブユニットを発現する能力はまた、異種ＰＫＳ酵素が本発明の包括的な過剰産生宿主細胞中で産生され得ることを示している。
【００３７】
前駆体指向型生合成は、ＤＥＢＳのモジュール１のケトシンターゼ（ＫＳ１）に変異を導入し、そしてジケチド前駆体の化学的に合成されたＮ−アセチルシステアミン（ＮＡＣ）チオエステルを発酵物に添加することによって、Ｓｔｒｅｐｔｏｍｙｃｅｓ ｃｏｅｌｉｃｏｌｏｒ中で、６−ｄＥＢの多くのＣ−１３誘導体を作製するために使用される。前駆体指向型生合成によるエリスロマイシンアナログの産生を単純化するためおよびポリケチドからのエリスロマイシンＡ類似体への産生を完全にプロセスするために、株Ｋ３９−１４を作製するＳａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ過剰産生株の染色体中にＫＳ１°変異を導入し、これを、図３中に示すプロピルＮＡＣジケチド（４）またはビニルＮＡＣジケチド（５）０．２５ｇ／Ｌの存在下、Ｆ１培地で発酵させた（本明細書中で参考として援用される、Ｂｒｕｎｋｅｒら，１９９８，Ｍｉｃｒｏｂｉｏｌ．１４４：２４４１〜２４４８を参照のこと）。この株は、各々、対応する１５−メチルエリスロマイシンまたは１４，１５−デヒドロエリスロマイシン誘導体（図２の化合物６〜８および９〜１１）を１０〜２０ｍｇ／Ｌの間で産生した。両エリスロマイシンアナログはＢａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓに対して抗菌活性を示した。１５−メチルエリスロマイシンＡの構造は、ＮＭＲによって決定した。
【００３８】
エリスロマイシンの量は、おそらく膜移送障壁のために、この株の潜在的な能力よりもかなり低いものであるが、これらの収率は小培養量からの生物学的スクリーニングには充分である。化学的に合成され得る合成ジケチドの可能な配列が与えられれば、簡便な様式でより多くのエリスロマイシン誘導体を生成し、スクリーニングすることが可能である。
【００３９】
本発明の方法に基づき、天然では株によって主生成物として産生されない種々のエリスロマイシン化合物を産生するために、Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａの過剰産生株を使用する。Ｓ．ｅｒｙｔｈｒａｅａ過剰産生株におけるＫＳ１ ヌル変異を含むＤＥＢＳ１変異株のＯｌｅＡＩ相補は、２種の異なるＰＫＳ酵素複合体から誘導されるタンパク質からなる機能的異種ＰＫＳであって未修飾の過剰産生株によっては主生成物として作製されないポリケチドを産生するＰＫＳの例を提供する。Ｓ．ｅｒｙｔｈｒａｅａ過剰産生株における１０，１１−アンヒドロエリスロマイシンの産生は、２種の異なるＰＫＳ酵素から誘導される酵素ドメインからなるモジュールを含むタンパク質を含む機能的異種ＰＫＳ複合体であって未修飾の過剰産生株によっては作製されないポリケチドを産生するＰＫＳ複合体の例を提供する。ＫＳ１ヌル変異を含むＳ．ｅｒｙｔｈｒａｅａ過剰産生株におけるジケチド補給によるポリケチドの産生は、ＰＫＳのための合成化学スターターを提供することによる野生型ＰＫＳによって産生されるポリケチドと異なるポリケチドを産生するために使用され得る非機能的異種ＰＫＳ複合体の例を提供する。
【００４０】
従って、本発明は組換え宿主細胞において多くの異なるポリケチドを産生するための有用な方法および宿主細胞を提供する。しかし、高い力価のポリケチドを産生するために変異誘発を経て最適化されたポリケチド産生宿主細胞が形質転換されにくいことは公知である。さらに、このような細胞で観測される形質転換の低い効率は、細胞を形質転換するために使用されるベクターのサイズが増加するにつれて顕著に減少する。非常に大きなベクターでは、形質転換は、しばしば単純に可能ではない。通常ＰＫＳ遺伝子が非常に大きい（＞３０ｋｂ）ために、この低い形質転換効率は、本発明の利点を実現するのに顕著な障壁となる。この形質転換障壁は宿主における制限−修飾系によるものであり得るといういくつかの証拠および推測がある。
【００４１】
接合媒介性の形質転換は、この障壁をある程度克服し得る；これは制限−修飾系への接合において生じる単鎖ＤＮＡのより低い感受性の結果であり得る。しかし、接合媒介性の形質転換であっても、相同組換えによって宿主細胞の染色体内に組み込まれる非常に大きなベクターでは成功し得ない。この障壁は相同組換え事象の性質の結果であり得、制限−修飾系によって送達されたＤＮＡの開裂と比較すると低くあり得る。
【００４２】
ファージからの結合部位（ａｔｔＰ）を含むベクター（例えば、ｐｈｉＣ３１、ｐＳＡＭ、およびｐＳＬＰベクター系のベクター）は、ファージのインテグラーゼ酵素によって媒介される機構によって宿主細胞の染色体に組み込まれ得る。しかし、この種のベクターがＳａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ宿主細胞での接合媒介性の形質転換に使用された場合、上述のように非常に低い形質転換効率が観測された。接合完了体（ｅｘｃｏｎｊｕｇａｎｔ）は、ＤＮＡ配列分析によって分析され、ベクターが、他の生物中のファージによって利用され、そして以前確認された好ましい結合部位の配列（ａｔｔＢ配列）とは配列において異なる部位に無作為に組み込まれたことを発見した。この低い形質転換効率の障壁は、ａｔｔＢ配列を含むように宿主細胞の染色体を変化させることによって克服された。得られた組換え宿主細胞は、ＰＫＳ遺伝子クラスター全体を含む非常に大きなベクターとであっても、ｐｈｉＣ３１結合部位配列を含むベクターとの接合によって、比較的高い形質転換効率で形質転換され得る。
【００４３】
従って、ある実施形態において、本発明はＥ．Ｃｏｌｉまたはヒト宿主細胞以外の組換え宿主細胞を提供し、この組換え細胞は、この細胞が誘導される細胞と比較して１以上のａｔｔＢ部位を含むように改変されている（本明細書中で参考として援用される、Ｔｈｏｒｐｅら，１９９８，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ９５：５５０５〜５５１０，およびＧｒｏｔｈら，２０００，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ９７：５９９５〜６０００を参照のこと）。複数の結合部位を含むように修飾された細胞は、染色体の異なる場所に複数の遺伝子を導入する目的のために好ましくあり得る。１つの実施形態において、変異誘発によって最適化された細胞からこの細胞が誘導され、高レベルでポリケチドを産生する。別の実施形態において、この細胞は、ＮＲＲＬ２３３８のようなＳａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ宿主細胞またはエリスロマイシンを２．５ｇ／Ｌを越えて産生する細胞である。
【００４４】
別の実施形態において、本発明は、ｐｈｉＣ３１結合部位を含むように改変された細胞の形質転換体を得るための方法を提供し、この方法は、前述の細胞と、相補的な結合部位（ａｔｔＢとａｔｔＰは互いに相補的である）を含むベクターを含む細胞とをインテグラーゼ（これはｐｈｉＣ３１インテグラーゼであり得、従って遺伝子が、ベクター中または宿主細胞中にあり得、そして異種プロモーターの制御下であり得る）存在下で、接合する工程を包含する。一方ａｔｔＰは、典型的にベクターおよび染色体上のａｔｔＢ上で使用されるが、一つはまた本発明の方法に従って、染色体中にａｔｔＰをそしてベクター上にａｔｔＢを配置し得る。別の実施形態において、本発明はこのような方法によって産生された細胞を提供する。好ましい実施形態において、このような方法によって産生された細胞は、前述の接合工程の前に細胞によって産生されないポリケチドを産生する。
【００４５】
以下の実施例は本発明を説明する目的のために与えられ、本発明または特許請求の範囲を限定するものとして解釈すべきではない。
【００４６】
（実施例１）
（株、培地条件、およびＤＮＡ操作）
ＤＮＡ操作はＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＸＬ１−Ｂｌｕｅ（Ｓｔｒａｔａｇｅｎｅ）中で行なった。Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ過剰産生株は、エリスロマイシンを７ｇ／Ｌを越えて産生した（株Ｋ１）。この株の形質転換を、ＹＥＭＥの代わりにＴＳＢ培地を用いた以外は、標準Ｓｔｒｅｐｔｏｍｙｃｅｓプロトプラスト法に従って行った。形質転換体は、Ｒ２ＹＥ再生プレートまたはＲ５再生プレート上のチオストレプトン（ｔｈｉｏｓｔｒｅｐｔｏｎ）（５０μｇ／ｍＬ）またはアプラマイシン（ａｐｒａｍｙｃｉｎ）（１００μｇ／ｍＬ）とともに選別した。
【００４７】
（実施例２）
（Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ ＫＳ１ヌル変異株の構築）
プラスミドｐＫＯＳ４０−５７を、ＤＥＢＳ ＫＳ１ヌル変異体（アラニンとＤＥＢＳ ＫＳ１ドメインの活性部位システインとの特殊な置換；本明細書中で参考として援用される、Ｋａｏら，１９９６，Ｂｉｏｃｈｅｍｉｓｔｒｙ ３５：１２３６３〜１２３６８を参照のこと）を含む４．０ｋｂ ＮｄｅＩ−ＥｃｏＲＶフラグメントを、送達ベクターとしての自殺ベクター（アプラマイシン耐性付与遺伝子を含むｐＵＣ１８誘導体）に導入することによって構築した。プラスミドｐＫＯＳ４０−５７は、Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ Ｋ１に形質転換された。アプラマイシン耐性株 （Ｋ１−１）をアプラマイシン（１００μｇ／ｍｌ）を含むＴＳＢ培地内で増殖させて、耐性を確認し、そして組み込みを染色体ＤＮＡのＰＣＲ分析によって確認した。二重交差決定のために、形質転換株をＴＳＢ倍地中でアプラマイシンなしで２回継代した。プロトプラストをＲ２ＹＥプレート上で再生した。再生プレートで生じる個々のコロニーを、次いで、アプラマイシンへの感受性についてスクリーニングした。所望の変異を含むコロニーは、エリスロマイシンを産生しないことが実証され、Ｓ．ｅｒｙｔｈｒａｅａ Ｋ３９−１４と命名された。上述のように、ｏｌｅＡＩ遺伝子をコードするプラスミドｐＫＯＳ３９−１１０と形質転換した場合、Ｋ３９−１４株は１５−ノルエリスロマイシンを産生し、そしてプロピルＮＡＣジケチド（図３の化合物４）を与えた場合、１５−メチル−エリスロマイシンを産生した。
【００４８】
（実施例３）
（エリスロマイシン誘導体の産生および分析）
本明細書中で記載されるＳａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ株を、Ｒ２ＹＥ ２５ｍＬ中で３０℃で５日間増殖させた。培養物を、エリスロマイシン誘導体についてＨＰＬＣ／ＭＳによって分析した。構造決定は、操作された株に関して予測された構造とマススペクトルとＨＰＬＣプロフィールとの間の一致に主として基づいた。マススペクトルおよびＨＰＬＣに関して、Ｓ．ｅｒｙｔｈｒａｅａ野生型株を参照として使用した。また、実施例４に記載されるように、１５−メチルエリスロマイシンＡのＮＭＲスペクトルによってさらなる構造確認が行なわれた。生物活性を、指標菌としてのＢａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓを用いた寒天プレート拡散アッセイによって評価し、阻害領域を視覚化するために３７℃で一晩中インキュベートした。
【００４９】
（実施例４）
（１５−メチルエリスロマイシンＡの特性決定）
Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ株Ｋ３９−１１４を、実施例３に記載されるように、発酵開始２４時間後に培養物に与えられた０．２５ｇ／ＬのプロピルＮＡＣジケチド（図３の化合物４）存在下で、発酵させた。発酵から抽出された１５−メチルエリスロマイシンＡを、特性決定のためにトルエン／アセトン １：１を用いるカラムクロマトグラフィーによってさらに精製して、白色固体を得た。^１Ｈおよび^１３Ｃスペクトルを、４００ＭＨｚ（Ｂｒｕｋｅｒ ＤＲＸ）でＣＤＣｌ_３溶液中で記録した。薄層クロマトグラフィーをシリカゲル６０でプレコートされたガラス上で行い、そしてスポットをバニリン染色によって視覚化し、次いで、加熱した。ＮＭＲ値：^１Ｈ ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３）δ ５．１４（ｄｄ，１Ｈ，Ｊ＝２．０，８．８），４．９１（ｄ，１Ｈ，Ｊ＝４．８），４．４４（ｄ，１Ｈ，Ｊ＝７．２），４．０５−３．９８（ｍ，２Ｈ），３．８４（ｂｒ ｓ，１Ｈ），３．６０（ｄ，１Ｈ，Ｊ＝７．６），３．５４−３．４７（ｍ，１Ｈ），３．３４（ｓ，３Ｈ），３．２５（ｄｄ，１Ｈ，Ｊ＝７．２，１０．４，），３．１２（ｂｒ ｑ，１Ｈ，Ｊ＝７．０），３．０４（ｄ，１Ｈ，Ｊ＝９．６），２．９０（ｄｑ，１Ｈ，Ｊ＝７．２，９．１），２．７７−２．６７（ｍ，１Ｈ），２．５２（ｄｄｄ，１Ｈ，Ｊ＝４．４，９．６，１２．４），２．４３−２．３７（ｍ，１Ｈ），２．３４（ｓ，６Ｈ），２．０５−１．９０（ｍ，２Ｈ），１．９０−１．８０（ｍ，２Ｈ），１．７９−１．５５（ｍ，３Ｈ），１．５０（ｓ，３Ｈ），１．３６−１．０９（ｍ，２７Ｈ），０．９４（ｔ，３Ｈ，Ｊ＝７．２）。^１３Ｃ ＮＭＲ（１００ＭＨｚ，ＣＤＣｌ_３）δ ２２１．２，１７４．６，１０２．２，９５．４，８２．５，７９．０，７７．０，７６．３，７６．０，７４．１，７３．５，７１．６，６９．９，６８．０，６４．６，６４．５，４８．５，４４．２，４３．９，３９．２，３８．５，３７．６，３６．８，３４．０，２９．２，２７．８，２６．１，２０．５，２０．４，１８．５，１７．７，１７．３，１５．２，１４．９，１３．０，１１．０，８．１。
【００５０】
（実施例５）
（ネイティブなＰＫＳ遺伝子を欠失している包括的産生宿主）
Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ高プロデューサの収量を向上する多くの変異がＰＫＳ遺伝子以外の染色体領域に存在し、前駆体プール、調節プロセス、栄養の利用、そして他の要因に影響を及ぼす。このような収量を向上する変異は、変異体および異種ＰＫＳ遺伝子によってコードされたポリケチドの過剰産生のための包括的な宿主として株を使用することによって、直接的および速やかに開発され得る。このことは主に、特定のポリケチドを作製するためにＰＫＳおよび補酵素によって使用される全ての出発物質を供給する能力を要求する。運良く、モジュラーＰＫＳは全て、単純なカルボン酸の１以上の小さな基（例えば、アセテート、プロピオネートまたはブチレートならびにこれらの２−カルボキシ誘導体）を使用して、炭素骨格およびジデオキシヘキソースまたはトリデオキシヘキソース（主要なポリケチドのグリコシドを形成するための１−チミジンジホスホグルコースから作製される）を構築する。他のテーラーリング（ｔａｉｌｏｒｉｎｇ）反応は全て、ほとんどの細菌性細胞中で利用可能な共通の補助因子を含む。従って、Ｓ．ｅｒｙｔｈｒａｅａは、出発物質の全てまたはほとんどを提供し得る；特定のポリケチドにとって必要とされる特殊な出発物質のいくつかは、遺伝子操作によって導入される。
【００５１】
Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａエリスロマイシン過剰産生体由来のＤＥＢＳ遺伝子を、非過剰産生株由来の異種ＰＫＳで交換し得る。導入されたＰＫＳ遺伝子によって作製された代謝産物の増強された産生は、他のポリケチドの産生のために開発され得る代謝産物過剰産生のための因子を宿主が実際に含むことを実証し得る。異種遺伝子をＳ．ｅｒｙｔｈｒａｅａ過剰産生株に導入する前に、ネイティブのＤＥＢＳ遺伝子を最初に欠失し得る。以下にさらに詳細に記載されているように、これは２段階相同組換え手順によって達成された。ＤＥＢＳ遺伝子に隣接する配列（約１．５ｋｂ）は、ＰＣＲによって増幅され、そして選択マーカー（アプラマイシン耐性のための）を有する自殺ベクターにクローン化された。この株を、プラスミド、および選択可マーカーを用いることによって選択された単一交差を用いて形質転換した。コロニーを選択せずに増殖させ、そして二重交差組換えを示すアプラマイシン耐性の損失についてスクリーニングした。二重交差候補の遺伝子型を、ＰＣＲを用いた分析、エリスロマイシン産生の損失、およびサザンブロットハイブリダイゼーションによって確認した。このように確認された株の一つをＫ９７−７１と命名された。
【００５２】
Ｓ．ｅｒｙｔｈｒａｅａ Ｋ９７−７１は３種のｅｒｙＡ遺伝子の染色体欠失およびＰｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ由来のｘｙｌＥ遺伝子の挿入を染色体中の適所に含む。この株を作製するために、プラスミドｐＫＯＳ９７−４９ｂを最初に以下のように構築した。ｅｒｙＡ遺伝子をフランキングした２種のフラグメントは、Ｓ．ｅｒｙｔｈｒａｅａゲノムＤＮＡから以下のようなプライマーを使用してＰＣＲ増幅した（ＳｐｈＩ、ＨｉｎｄＩＩＩ、ＢａｍＨ ＩおよびＥｃｏＲＩ制限部位は下線が引かれている）：
ｅｒｙＡＩ左フランク順方向：
５’−ＴＴＴＧＣＡＴＧＣＧＧＣＣＡＣＧＣＧＣＡＣＧＴＣＧＴＧＡＣＣ，
ｅｒｙＡＩ左フランク逆方向：
５’−ＴＴＡＡＧＣＴＴＣＡＴＡＴＧＴＣＣＣＣＣＴＡＣＴＣＧＡＣＧＡＣＣＡＣ；
ｅｒｙＡＩＩＩ右フランク順方向：
５’−ＴＴＴＧＧＡＴＣＣＧＧＣＧＧＡＧＧＧＡＡＴＡＣＡＴＧＡＣＣＡＣＧＡＣ，
ｅｒｙＡＩＩＩ右フランク逆方向：
５’−ＴＴＴＧＡＡＴＴＣＣＣＧＣＴＣＧＣＴＧＡＡＧＴＣＣＡＧＧＣＴ。
次いで２種のフラグメントを、下線を引かれた制限部位およびｐＳＥＴ１５２中の対応する部位を用いてｐＳＥＴ１５２内にクローン化した(本明細書中に参考として援用される、Ｂｉｅｒｍａｎら，１９９２，Ｐｌａｓｍｉｄ ｃｌｏｎｉｎｇ ｖｅｃｔｏｒｓ ｆｏｒ ｔｈｅ ｃｏｎｊｕｇａｌ ｔｒａｎｓｆｅｒ ｏｆ ＤＮＡ ｆｒｏｍ Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ｔｏ Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐｐ．，Ｇｅｎｅ １１６：４３〜４９を参照のこと）。得られたプラスミドはｐＫＯＳ９７−４９ａと命名された。
ｘｙｌＥ遺伝子をＰＣＲ増幅して、ｐＫＯＳ−９７−４９ａのＮｄｅＩおよびＸｂａＩ部位にクローン化して、ｐＫＯＳ９７−４９ｂを作製した。このプラスミドはもはやｐｈｉＣ３１インテグラーゼ遺伝子およびｐＳＥＴ１５２由来のａｔｔＰ遺伝子座を含んでおらず、そして相同組換えによる送達のための自殺ベクターとして役立つ。
【００５３】
過剰産生株Ｓ．ｅｒｙｔｈｒａｅａ Ｋ１のプロトプラストは、細胞をＴＳＢ培地中で成長さ、そして成長３日後に収集することを除いて、標準手順によって生成した（本明細書中に参考として援用される、Ｈｏｐｗｏｏｄら，１９８５，Ｇｅｎｅｔｉｃ Ｍａｎｉｐｕｌａｔｉｏｎ ｏｆ Ｓｔｒｅｐｔｏｍｙｃｅｓ：Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ．Ｔｈｅ Ｊｏｈｎ Ｉｎｎｅｓ Ｆｏｕｎｄａｔｉｏｎ，Ｎｏｒｗｉｃｈ，ＵＫを参照のこと）。プラスミドｐＫＯＳ９７−４９ｂ ＤＮＡは、Ｅ．ｃｏｌｉ ＥＴ１２５６７から調製して、非メチル化ＤＮＡを生成して、ＰＥＧ３３５０（Ｓｉｇｍａ）を使用する以外は標準手順を使用するプロトプラストの形質転換に使用した（上述のＨｏｐｗｏｏｄらの文献を参照のこと）。プレートはアプラマイシン（ａｐｒａｍｙｃｉｎ）１ｍｇで覆って、相同組換えによるプラスミドの組込みのために選択した。アプラマイシン耐性コロニーはサザンハイブリダイゼーションによって分析し、予期される位置での単一交差を確認した。以下に示す二重交差を実行するために、１つのコロニーを選択した。株はＩＴ寒天培地（１Ｌあたり、５ｇ グルコース、５ｇ トリプトン、０．５ｇ ベタイン塩酸塩、５ｇ コーンスターチ、１ｇ コーンスティープ液（ｃｏｒｎ ｓｔｅｅｐ ｌｉｑｕｏｒ）（５０％）、２００ｍｇ ＭｇＳＯ_４・Ｈ_２Ｏ、２ｍｇ ＺｎＳＯ_４＿７Ｈ_２Ｏ、０．８ｍｇ ＣｕＳＯ_４＿５Ｈ_２Ｏ、０．２ｍｇ ＣｏＣｌ_２＿６Ｈ_２Ｏ、４ｍｇ ＦｅＳＯ_４＿７Ｈ_２Ｏ、８０ｍｇ ＣａＣｌ_２＿６Ｈ_２Ｏ、１５０ｍｇ ＫＨ_２ＰＯ_４、 １０ｇ ＮａＣｌ、２０ｇ 寒天）上で十分に胞子成長するまで、非選択に成長した。胞子を収集し、ＩＴプレート上に希釈して、そして単一コロニーをアプラマイシン耐性の欠損についてスクリーニングした。エリスロマイシンを産生しない１つのアプラマイシン感受性のコロニーを単離した。サザンハイブリダイゼーションによって二重交差を確認した。この株をＳ．ｅｒｙｔｈｒａｅａ Ｋ９７−７１と命名した。
【００５４】
株Ｋ９７−７１は、他の非６−ｄＥＢポリケチドについてのＰＫＳ遺伝子を挿入し得る本発明の包括的な過剰産生宿主である。それらの大きなサイズ（３０ｋｂまたはそれ以上）に起因して一度にＰＫＳ遺伝子の完全なセットを移動することが困難であり得るので、その構築は必要であれば一連の２〜３の二重相同組換え工程に分けられ得る。ピクロマイシン（ｐｉｃｒｏｍｙｃｉｎ）ＰＫＳ遺伝子は本発明のこの面の実例として役立つ。ピクロマイシンＰＫＳはＤＥＢＳに関するが、顕著に異なる化合物を産生する。ピクロマイシンＰＫＳは、ナルボノリド（ｎａｒｂｏｎｏｌｉｄｅ）（炭素１０のメチル基、炭素１０と１１間に二重結合、および炭素３のケトンがないこと以外は６−ｄＥＢと類似）を合成する。これらはピクロマイシンＰＫＳのモジュール２およびモジュール６における違いに起因している。ＰＫＳのモジュール２のＡＴドメインはプロピオナートの代わりにアセテートを組み込み、モジュール２においてさらなるデヒドラターゼ（ＤＨ）活性があって、モジュール６におけるケトレダクターゼ（ＫＲ）ドメインがない。また、ピクロマイシンおよびＤＥＢＳ ＰＫＳの構築は、ピクロマイシンのモジュール５およびモジュール６が２種のＯＲＦに分かれている以外は類似している。２種のＰＫＳ間のアミノ酸配列の類似性は約４０％である。同時に、ピクロマイシンＰＫＳのこれらの特徴的な特徴は、ピクロマイシンＰＫＳをエリスロマイシン過剰産生生物に移入される場合、ＤＥＢＳと異なるモジュラーＰＫＳがポリケチドを過剰産生することを説明するための理想的な選択にする。
【００５５】
異種ＰＫＳ遺伝子を導入するために、ａｔｔＰ結合部位を含むプラスミドを使用し得る。あるいは、上述の二重組換え手順を使用し得る。一旦完全なＰＫＳが導入されると、株は代謝産物の産生のために分析される。エリスロマイシンデオキシ糖生合成遺伝子およびグリコシルトランスフェラーゼ遺伝子がインタクトなままであるため、ｅｒｙ遺伝子クラスター中に存在するグリコシルトランスフェラーゼの活性に依存して、ポリケチドアグリコン（すなわち、ナルボノリド）、デソスアミニル化（ｄｅｓｏｓａｍｉｎｙｌａｔｅｄ）誘導体（ナルボマイシン）のいずれかまたは両方が産生され得る可能性がある。さらに、Ｃ−６またはＣ−１２ヒドロキシル化反応のいずれかが起こり得る。親株中の、産生するポリケチドの合計量は、過剰産生を示すための同様の条件下でのエリスロマイシンＡの産生水準と直接的に比較される。
【００５６】
他のＰＫＳ遺伝子は容易に株Ｋ９７−７１メガロマイシン（ｍｅｇａｌｏｍｉｃｉｎ）ＰＫＳ遺伝子内に導入され得る（本明細書中に参考として援用される、ＰＣＴ特許出願番号 ＵＳ００／２７４３３、２０００年１０月４日出願を参照のこと）および、オレアンドリド（ｏｌｅａｎｄｏｌｉｄｅ）ＰＫＳ遺伝子（本明細書中に参考として援用される、ＰＣＴ公開番号 ００／０２６３４９を参照のこと）を含むがこれらに限定されない。また、この宿主は、ＦＫ５０６およびエポシロン（ｅｐｏｔｈｉｌｏｎｅ）のような、エリスロマイシンに遠くにのみ関係したポリケチドの産生のために使用され得る。これはまた、ＰＫＳ酵素に対する独特の基質（例えば、２−エチルマロネートまたは２−ヒドロキシマロネート）の産生を確実にするか、またはそれが形成されるとき、ポリケチド骨格を修飾する（例えば、脱水または酸化）酵素を提供するために、特定の非ＰＫＳ遺伝子の導入を伴う。さらに、種々のプロモーター（例えば、エリスロマイシン過剰産生株からのａｃｔプロモーター、ｅｒｍＥ^＊プロモーター、ｅｒｙＡプロモーター）のうち任意のものを利用して、異種ＰＫＳ遺伝子クラスターの発現を駆動しうる。
（実施例６）
（ＰｈｉＣ３１結合部位を含むＳ．ｅｒｙｔｈｒａｅａ株の構築）
Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ株は、ｐｈｉＣ３１の結合部位配列に実際に対応する染色体のＤＮＡ配列を含まない。従って、ｐｈｉＣ３１から誘導されたプラスミドとの結合効率は、このような株とでは高くなく、このような株への非常に大きなプラスミドの導入は不可能ではないとしても、困難であり得る。結果として、本発明は、ポリケチド産生株の染色体ＤＮＡ内にｐｈｉＣ３１結合部位配列を挿入することによって改良された種々のポリケチド産生株（Ｓ．ｅｒｙｔｈｒａｅａ株を含むがこれに限定されない）を提供する。
【００５７】
Ｓ．ｅｒｙｔｈｒａｅａ Ｋ２４−１は３種のｅｒｙＡ遺伝子の染色体欠失ならびにＳｔｒｅｐｔｏｍｙｃｅｓ ｌｉｖｉｄａｎｓ由来のＳｔｒｅｐｔｏｍｙｃｅｓファージｐｈｉＣ３１のためのａｔｔＢ遺伝子座、次いでｅｒｍＥ^＊プロモーターの適所での挿入を含む。この株を作製するために、プラスミドｐＫＯＳ１３４−０４が最初に以下のように構築された。以下の２種のアニーリングされたオリゴヌクレオチドを使用して、ｐｈｉＣ３１ ａｔｔＢ部位をｐＫＯＳ９７−４９ａのＨｉｎｄ ＩＩＩとＢａｍＨ Ｉ部位との間に挿入した：
順方向：
５’−ＡＧＣＴＴＣＧＧＧＴＧＣＣＡＧＧＧＣＧＴＧＣＣＣＴＴＧＧＧＣＴＣＣＣＣＧＧＧＣＧＣＧＴＡＡ−ＣＴＡＧＴＧ、
および
逆方向：
５’−ＧＡＴＣＣＡＣＴＡＧＴＴＡＣＧＣＧＣＣＣＧＧＧＧＡＧＣＣＣＡＡＧＧＧＣＡＣＧＣＣＣＴＧＧ−ＣＡＣＣＣＧＡ。
このプラスミドは、ｐＫＯＳ０２４−８７と命名された。プラスミドｐＫＯＳ０１３４−０４は、ｐＫＯＳ０２４−８７の得られたＳｐｅＩとＢａｍＨＩ部位との間のｅｒｍＥ^＊プロモーターを含む約３００ｂｐのＮｈｅＩ／ＢａｍＨＩフラグメントを挿入することによって作製した。
【００５８】
プラスミドｐＫＯＳ０１３４−０４は、以下に記載するように、Ｅ．Ｃｏｌｉからの結合を経てＳ．ｅｒｙｔｈｒａｅａ Ｋ９７−７１に導入された。相同組換えによる単一交差および二重交差についてのスクリーニングを上述のように行なった。サザンハイブリダイゼーションによって確認された二重交差株が得られ、Ｓ．ｅｒｙｔｈｒａｅａ Ｋ２４−１と命名された。
Ｓ．ｅｒｙｔｈｒａｅａ株Ｋ９７−７１およびＫ２４−１は、１〜２のＩＴプレート上で増殖した株から胞子を収集することによって調製し、滅菌綿でこの胞子を濾過して、そして２０％グリセロール１ｍｌで再懸濁した。胞子懸濁液を約２０℃で保存した。胞子懸濁液２０μＬのアリコートを２×ＹＴ ５ｍｌに添加し、そして振盪しながら３０℃でインキュベートした。１時間後、胞子を遠心分離によって収集した（レシピエント細胞）。ドナー細胞を、Ｅ．ｃｏｌｉ／ｐＵＺ８００２をｐＳＥＴ１５２またはｐＫＯＳ９７−１１３またはｐＫＯＳ９７−６９ａを用いて形質転換し、そしてアプラマイシン耐性のみを選択することによって調製した。プラスミドｐＫＯＳ９７−１１３およびｐＫＯＳ９７−６９ａはそれぞれ、ｍｅｇＡＩ−ＩＩＩ遺伝子およびｅｒｙＡＩ−ＩＩＩ遺伝子を保有する約３０ｋｂ挿入物を含むｐＳＥＴ１５２の誘導体である。
【００５９】
一次形質転換プレートからいくつかのコロニーを選び、そしてクロラムフェニコール（１０μｇ／ｍＬ）、カナマイシン（１００μｇ／ｍＬ）、アプラマイシン（６０μｇ／ｍＬ）を含むＬＢ ５ｍｌに播種するために使用した。細胞を３７℃で３〜４時間増殖させ（０．４〜０．６のＯＤ６００）、遠心分離によって収集し、ＬＢ ５ｍｌで洗浄し、遠心分離して、そしてＬＢ １００μＬに再懸濁した。ドナー細胞とレシピエント細胞との間の接合移入は、ドナー懸濁液１００μＬにレシピエント細胞を再懸濁させ３７℃で１５〜３０分間振盪せずにインキュベートすることによって行なった。５０μｇ／ｍＬのナリジクス酸を含むＲ５プレート上に細胞を塗布し、３４℃で１６時間インキュベートした。次いでプレートをナリジクス酸１ｍｇおよびアプラマイシン２ｍｇを含む軟栄養寒天３ｍｌで覆った。接合完了体をさらに４８時間インキュベートした後に観測した。
【００６０】
接合頻度はおおよそ、Ｋ２４−１：：ｐＳＥＴ１５２で５×１０−５、Ｋ９７−７１：：ｐＳＥＴ１５２で３×１０−７、Ｋ２４−１：：ｐＫＯＳ９７−１１３で１×１０−６、およびＫ９７−７１：：ｐＫＯＳ９７−１１４で１×１０−７未満であった。
【００６１】
Ｋ２４−１：：ｐＳＥＴ１５２接合完了体およびＫ９７−７１：：ｐＳＥＴ１５２接合完了体の染色体へのプラスミド組み込みは以下のように確認した。ゲノムＤＮＡを、各株のいくつかの接合完了体から調製しＢａｍＨＩまたはＥｃｏＲＩと共に消化し、再連結し、そしてＥ．Ｃｏｌｉを形質転換するために使用した。アプラマイシンは染色体中に組み込まれたｐＳＥＴ１５２ベクターおよびａｔｔＢ／ａｔｔＰ接合配列を含む再び環状にしたＤＮＡを選択するために使用した。プラスミドＤＮＡをアプラマイシン耐性コロニーから調製し、ａｔｔＢ／ａｔｔＰ接合を、ｐＳＥＴ１５２中のａｔｔＰ部分の上流にアニーリングしたプライマーを使用して配列させた。分析された４種の別々のＫ２４−１：：ｐＳＥＴ１５２接合完了体において、ｐＳＥＴ１５２は４つの異なる染色体位置に組み込まれ、これらは、他のＳｔｒｅｐｔｏｍｙｃｅｓ ｓｐｐ．中の公知のａｔｔＢ部位との類似性を示さなかった。分析された７種のＫ２４−１：：ｐＳＥＴ１５２接合完了体において、ｐＳＥＴ１５２は、いずれの場合も操作されたｐｈｉＣ３１ ａｔｔＢに組み込まれた。
【００６２】
これらの結果は、インテグラーゼｐｈｉＣ３１が媒介するベクターの組み込みが、操作されたａｔｔＢ部位が存在しないＳ．ｅｒｙｔｈｒａｅａ ｓｐｐ．において部位特異的ではなく、比較的低い頻度で起こることを示している。染色体中での適切なａｔｔＢ部位またはａｔｔＰ部位の配置は、この実施例で示した一例のように、ｐｈｉＣ３１をベースとするベクターの接合頻度が顕著に増加し、結果として部位特異的組み込みが起こる。
【００６３】
記載された説明および実施例によって、本発明が記述されるが、当業者は、本発明が種々の実施形態において実施され得ることを理解し、そして前述の記載および実施例は例示を目的とし、特許請求の範囲を限定するものではないことを理解する。
【図面の簡単な説明】
【図１】
図１は、ＤＥＢＳを構成する３種のタンパク質（ＤＥＢＳ１、ＤＥＢＳ２、およびＤＥＢＳ３）のモジュラー組織、６−デオキシエリスロノリドＢ（６−ｄＥＢ）の合成における各工程で形成される中間体、および６−ｄＥＢの修正から生じるエリスロマイシンの構造（エリスロマイシンＣは示されていない）を含む、エリスロマイシン生合成における重要な工程を示す。
【図２】
図２は、モジュール２のＫＲドメインと、ラパマイシンＰＫＳのモジュール４のＫＲおよびＤＨドメインとのＤＥＢＳ中での置換ならびに得られた組換えＰＫＳによって産生した生成物ならびにＳａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ中のポリケチド修飾酵素の作用、を示す。
【図３】
図３は、前駆体からの直接生合成によるエリスロマイシン誘導体の産生を示し、２種のジケチド（化合物４および５）およびＫＳ１°変異を含む本発明のＳａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ宿主細胞にこれらを提供して形成した生成物（化合物６〜１１）を示している。[0001]
(Technical field)
The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. The present invention relates to the technical fields of agriculture, veterinary science, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary medicine.
[0002]
(Background technology)
Polyketides are an important class of natural products that are responsible for the development of many human therapeutic, veterinary and agricultural products (eg, FK506, lovastatin, and avermectin). Enzymes that synthesize these compounds, polyketide synthases (PKS), are targets of various molecular engineering approaches aimed at producing either improved analogs of existing drugs or combinatorial libraries of novel polyketides. It has become. Modular PKS (eg, 6-deoxyerythronolide B synthase (DEBS) shown in FIG. 1) has a novel polyketide structure that is derived by genetic manipulation of one or more enzyme domains contained in this type of PKS enzyme. It has been modified by this type of technology to produce (see above, US Pat. No. 5,962,290 and PCT Publication No. 98/49315).
[0003]
Since these first generation successes, it has resulted in rapid growth of genetically engineered PKSs that produce new polyketides or “non-natural” natural products, each of which is incorporated herein by reference in PCT Publication No. 99. / 61599, 00/024907, and 00/026349). Recent research has led to the creation of libraries of about 100 macrocycles, indicating the possibility of creating libraries with significant complexity and diversity, each incorporated herein by reference. PCT Publication Nos. 00/063631 and 00/024907).
[0004]
The ability to manipulate the catalytic activity of these multifunctional enzymes as expected represents an important technical achievement in protein engineering. However, one of the current challenges to the construction of very large compound libraries (more than 1000 compounds) has been generated in connection with many genetically modified PKSs, particularly those with multiple domains modified The level is decreasing (previously, PCT Publication Nos. 00/063631 and 00/024907). Although it is desirable to use knowledge of the structure-activity of PKS to help guide more optimal operation of PKS, the current understanding is relatively limited due to the complexity and size of these enzymes. And progress is slow. Therefore, in order to advance combinatorial biosynthetic techniques, it is important to develop complementary approaches that do not rely on detailed understanding of the enzyme structure.
[0005]
One possibility impacts an important resource that has been generally served over the years against establishing a commercial process for producing natural product drugs. Almost inevitably, extensive cell line improvement and process development programs have been conducted to improve the yield of compounds from naturally occurring organisms, often achieving greater than a 100-fold increase in potency. Many microorganisms have been optimized through random mutagenesis for mass production of high-value compounds including penicillin, macrolide antibiotics, and lovastatin. Although this conventional approach to strain improvement can be applied to strains with engineered PKS, this approach is labor intensive, especially considering the many mutant strains that can be generated by combinatorial biosynthesis. Is a process. However, if the overproduction capacity of existing industrial strains can be applied to increase the potency of polyketides derived from engineered PKS, it represents an attractive and economical solution. Overproduction not only increases accessibility to compounds produced at levels that are currently too low for screening, but can also increase the size of the library produced. The reason is that the generation level became too low after more modifications were introduced into PKS. However, the broad applicability of this type of strain depends on what are the factors responsible for overproduction and whether production from recombinant PKS is increased in an overproduction environment.
[0006]
The present invention meets the need for a comprehensive host cell capable of producing polyketides at significantly higher levels than other host cells.
[0007]
(Summary of the Invention)
In a first embodiment, the present invention provides a method for producing a first polyketide, said method comprising producing a PKS producing said first polyketide in a cell optimized to produce a second polyketide. A method is provided that includes expressing an encoding polyketide synthase (PKS) gene.
[0008]
In one aspect, the method includes producing a first polyketide that is a derivative of a second polyketide, and modifying the PKS gene in the overproducing cell such that the gene expresses PKS that produces the first polyketide.
[0009]
In another aspect, the method includes the introduction of a gene expressing PKS that produces the first polyketide into an overproducing cell. In a preferred form, the gene expressing PKS producing the second polyketide is deleted or otherwise inactivated before or after introduction of the gene encoding the first PKS.
[0010]
In a preferred embodiment of this method, the overproducing cell produces a second polyketide at a level above 1 g / L, preferably above 2.5 g / L, more preferably above 5 g / L, and most Preferably, it produces exceeding 10 g / L. In a preferred embodiment of this method, the overproducing cells produce the first polyketide at a level above 10 mg / L, preferably above 25 mg / L, more preferably above 50 mg / L, and most preferably Produces over 100 mg / L.
[0011]
In another embodiment, the invention provides for the inclusion of the gene encoding the second polyketide being deleted from the cell, and the gene encoding PKS producing the first polyketide can be easily introduced and expressed in the cell. An overproducing host cell is provided.
[0012]
In one aspect, this global overproducing cell is derived from a Saccharopolyspora erythraea host cell that produces erythromycin at a level greater than 2.5 g / L and either by deletion of all or substantially all of the eryA gene or DEBS It is modified by either mutational inactivation of the ketosynthase (KS) domain of module 1 of PKS.
[0013]
In another embodiment, the present invention provides a global overproducing host cell that has been modified to express a PKS that produces a first polyketide. In a preferred embodiment, the host cell contains a binding site for the integrating phage phiC31 that facilitates transformation of the strain. In certain embodiments, the host cell is modified by genetic alterations to include this type of binding site.
[0014]
In one aspect, this over-producing host cell is derived from a Saccharopolyspora erythraea host cell that produces erythromycin at levels above 2.5 g / L and produces 10,11-anhydro-6-deoxyerythronolide B. It is modified either by altering the eryA gene to encode a PKS or by mutational inactivation of the ketosynthase (KS) domain of module 1 of DEBS PKS.
[0015]
These and other embodiments, modes, and aspects of the invention are described in more detail in the following description, examples, and appended claims.
[0016]
(Detailed description of the invention)
In a first embodiment, the present invention provides a method for producing a first polyketide, wherein the method described above produces a first polyketide in a cell that is optimized to produce a second polyketide. There is provided a method comprising expressing a PKS gene encoding (PKS). In a preferred embodiment of this method, the overproducing cell produces a second polyketide at a level greater than 1 g / L, preferably greater than 2.5 g / L, more preferably greater than 5 g / L, and most Preferably, it produces exceeding 10 g / L. In a preferred embodiment of this method, the overproducing cells produce the first polyketide at a level greater than 10 mg / L, preferably greater than 25 mg / L, more preferably greater than 50 mg / L, and most preferably Produces over 100 mg / L.
[0017]
Significant efforts to increase the production of polyketides in cells because the value of polyketides in pharmacy and agriculture, and the amount of polyketides produced in cells where they are found naturally are typically small Has been paid. Thus, there are many “overproducing” cells that make polyketides. However, by way of example only, erythromycin and semisynthetic derivatives have important pharmaceutical uses as anti-infective agents. DEBS (synthesizes 6-deoxyerythronolide B (6-dEB, compound 1 in FIG. 1), which is the macrocyclic lactone center of erythromycin) belongs to the modular class of PKS. The reason for being so called is that the enzyme domains required for synthesis are organized by modular repeats of the catalytic domain (FIG. 1). The organism from which DEBS was originally cloned, Saccharopolyspora erythraea NRRL 2338, typically produces erythromycin A between 100-200 mg / L in standard laboratory fermentations. Streptomyces coelicolor or S. no hydroxylation and glycosylation pathways are present therein. Heterologous expression of DEBS in any of the Lividans yields about 50-100 mg / L of 6-dEB (1) under optimal conditions, which is close to the amount of 1 produced by NRRL2338 (referenced herein) No. 5,672,491, incorporated by reference). In contrast, many industrially optimized S.I. The erythraea strain produces erythromycin in excess of 10 g / L.
[0018]
However, the biochemical reasons for this type of overproduction are not known. One possible reason is that compared to normal producer cells, overproducer cells have one or more mutations in the gene encoding PKS expression. Another reason is that overproducing cells simply produce more of the precursor compound used in polyketide biosynthesis. If the reason for overproduction is present in the PKS gene, it is unlikely that the overproducing cell of the first polyketide is the overproducing cell of the second polyketide.
[0019]
To demonstrate the advantages of the present invention, an overproducing strain of Saccharopolyspora erythraea named K1 capable of producing at least 7 g / L of erythromycin in bioreactor fermentation was used. As mentioned above, there are surprisingly few reports on the determinants of overproduction in industrial microorganisms. The increase in yield results from mutations in the biosynthetic enzymes that increase flux, the corresponding upregulation of gene expression, or higher intracellular retention of the substrate (acyl CoA thioester in the case of polyketides). S. For erythraea erythromycin overproducing cells, the most probable reason for the high production level in Saccharopolyspora erythraea overproducing cells remains the combination of the latter two reasons, and a significant increase in titer is simply the result of overexpression of DEBS It is unlikely to think. To demonstrate that the overproducing cell line did not carry a kinetically improved PKS, the PKS gene was cloned and expressed in Streptomyces lividans and Streptomyces coelicolor, and production levels were reduced to wild-type S. coli. Comparison was made with DEBS isolated from the erythraea strain. This result suggests that mutations in PKS are unlikely to be responsible for overproduction, and that expression of PKS from other organisms in this strain leads to higher titers compared to the parent strain. Suggested.
[0020]
To examine whether some or all of the improved production characteristics of the industrial Saccharopolyspora erythraea host were due to the whole or part of the activity of PKS, PKS was compared to DEBS from NRRL 2338 strain. Furthermore, since this industrial strain is produced from multi-stage mutagenesis, it is possible to obtain higher biosynthesis throughput as a result of the introduction of mutations in the DEBS gene. If so, Streptomyces coelicolor or S. By using a mutated PKS gene in lividans, it can be a strain that increases the titer of erythromycin and related macrolides produced in this type of heterologous host previously produced with DEBS. The complete set of DEBS genes from overproducing strains is sequenced to identify potential mutations, but the size of the gene cluster (> 30 kb) makes this approach difficult.
[0021]
Instead, three genes encoding DEBS, eryAI-III, were cloned from overproducing strains using conserved recognition sites and S. cerevisiae. Placed in a similar Streptomyces expression vector used for heterologous expression of DEBS obtained from erythraea NRRL2338. The resulting plasmid, pKOS108-04, is consistent with pKAO127'kan '(including wild type DEBS) except for the source of the eryAI-III gene. In either case, gene expression is controlled by the actI promoter and actII-4 regulatory protein on the expression plasmid (see US Pat. No. 5,672,491, incorporated herein by reference). . Both pKOS108-04 and pKAO127 'kan' were lividans K4-114 and S. Coelicolor CH999 was used to convert. Multiple transformants (each 2-4) were grown in parallel and the 6-dEB production profile was determined. S. lividans and S. In both Coelicolor, two different S. coli. The DEBS gene from the erythraea strain produced an equivalent amount of 6-dEB (approximately 30-40 mg / L) under the culture conditions described in the examples below.
[0022]
The inability to increase polyketide titers with PKS derived from overproducing hosts is that DEBS from this strain is not catalytically different from wild-type DEBS, but 6-dEB production levels are utilized in these strains. It implies the possibility that it is limited by the amount of possible precursors. This experimental result highlights the potential advantages of an industrial strain as an overproducing host than its use of PKS in a heterologous host. Thus, one advantage of the present invention is understood when an overproducing strain for one polyketide is genetically altered to express a PKS gene for a different polyketide.
[0023]
In one aspect, the method includes production of a polyketide that is a derivative of a second polyketide and alteration of the PKS gene in an overproducing cell, such as those genes that express PKS producing the first polyketide. This aspect of the invention is illustrated by an erythromycin overproducing strain of Saccharopolyspora erythraea optimized for combinatorial biosynthesis. A mutant of DEBS was constructed in this host by catalytic domain substitution that alters the structure of the corresponding polyketide. Compared to the expression of a similar mutant DEBS expressed in Streptomyces lividans (which is a non-optimized host), the production of modified polyketides was significantly higher.
[0024]
Many strategies for manipulating the biosynthesis of polyketides have been achieved using DEBS and other modular PKS. These include domain and module inactivation, deletion and substitution, construction of hybrid modules by protein fusion, and complementation between PKS subunits from different sources. These experiments are described in Streptomyces coelicolor, S .; lividans, or S. It is carried out in any of the relatively low production strains of erythraea. In accordance with the present invention, the potency of polyketides produced by the PKS strategy commonly used to modify DEBS in these strains can be substantially enhanced in overproducing strains.
[0025]
To illustrate this advantage, a representative PKS domain substitution was constructed in an overproduced Saccharopolyspora erythraea strain. Exchange of ketoreductase domains in DH and KR domains (rapDH / KR4) from DEBS augmentation module 2 (eryKR2) and rapamycin PKS augmentation module 4 in the chromosome of an overproducing host by homologous double recombination Was designed (FIG. 2). This particular mutation was selected to rigorously test its ability to improve low yields of PKS, with the lowest production of functional single mutants produced in systemic screening for domain substitution. is there. When modified in a plasmid-based DEBS system and expressed in Streptomyces lividans, the resulting strain (S. lividans K4-114 / pKOS11-64) is about 0.3 mg / L as a result of newly introduced DH activity. The 10,11-anhydro analog 3 of 6-dEB was produced (FIG. 2). (See both PCT Publication Nos. 98/49315 and 00/02497, incorporated herein by reference)
Saccharopolyspora erythraea strain K39-10 (Table 1) containing substitutions produced a mixture of large amounts of erythromycin analogues (which all contained the expected 10,11-anhydro moiety but differed in the hydroxylation pattern) (Figure 1). 2). The most abundant products were 10,11-anhydroerythromycin A (compound 4), B (compound 5), and D (compound 6), and 6-deoxy-10,11-anhydroerythromycin A (compound 7). ), B (Compound 8), and D (Compound 9). To confirm the structures of compounds 4, 5, 7, and 9 and to determine the position of the hydroxyl groups of compounds 5 and 7, ¹ H-NMR spectroscopy and ¹ H-TOCSY correlation was used. The ratio of these produced compounds can be media dependent. The B (12-deoxy) and D (4 ″ -desmethyl) species of erythromycin A are often S. cerevisiae. Although present in the fermentation broth of Erythraea strains, 6-deoxy compounds are usually not detected. EryF is cytochrome P450 that causes hydroxylation at the C-6 carbon of polyketide aglycone. The presence of a 6-deoxy compound at K39-10 is consistent with previous observations that the in vitro hydroxylase activity of EryF is poor for substrates with 10,11-double bonds.
[0026]
The total yield of erythromycin from either R5 fermentation medium or F1 fermentation medium was about 50 mg / L. This corresponds to approximately 26 mg / L of the macrocyclic lactone product (compound 3) synthesized by the modified PKS. Although the yield of polyketide was significantly reduced compared to the amount of erythromycin produced by the parent strain, there is an almost 100-fold increase in potency when compared to the production of compound 3 in Streptomyces lividans. This relative increase is consistent with the relative increase in 6-dEB production from unmodified DEBS in two different hosts, and most or all of the benefits of overproduction are translated into the mutant PKS It has been demonstrated that
[0027]
These experiments, which demonstrate that the use of high polyketide producing strains is a general approach to the construction of large polyketide combinatorial libraries, yielded two important findings. The first is that the production level derived from the mutant strain PKS can be significantly increased when expressed in an overproduction environment. This is shown in examples with domain substitutions that yield a relatively small amount of compound compared to many other single variants that are made. Thus, this type of increase in yield is probably universally observed, including strains containing PKS enzymes with multiple modifications compared to the wild type. Using an efficient genetic tool for this strain, an existing macrolide library is screened and the appropriate compound titers are generated to expand the number of available compounds. It is relatively convenient to move into the erythraea.
[0028]
These experiments also indicate that the cause of overproduction in this Saccharopolyspora erythraea strain is due to the host environment and not from mutations in the PKS gene. This also means that a module or domain from a heterologous PKS will produce a higher titer in the overproducing host. This is important. This is because the functional unit for PKS manipulation is available from many PKSs cloned from different organisms. Moreover, complete pathways for low production PKS (eg, pathways for anti-tumor polyketide epothilone) require sufficient substrate available in the strain and thereby need to improve upon the traditional strain It suggests that avoiding sex can be improved by heterologous expression.
[0029]
Thus, polyketide overproducing strains can be used to produce PKS-derived polyketide derivatives designed in good quantities. As indicated in the specification, the use of industrial erythromycin producing strains leads to the production of erythromycin derivatives in sufficient quantities for biological assays from small scale production cultures (<1 L) and at moderate scales. Up (100-1000 L) leads to the production of sufficient amounts of erythromycin derivatives to guide optimization by chemical modification. Thus, the present invention achieves a “total” increase in titers from polyketide libraries prepared by combinatorial biosynthetic strategies that translate the ability to create larger compound libraries in good yield. Provides a simple and experimental method. Furthermore, because identification and cloning of polyketide gene clusters can be accomplished in a relatively small amount of time, it is possible to significantly reduce the process development time and efforts to derive polyketides by heterologous expression in overproducing hosts. Furthermore, it is possible to reverse the reduced potency of modified PKS-derived polyketides by using a host optimized for very high production levels.
[0030]
The methods of the present invention can include not only alteration of existing PKS genes in overproducing cells, but also introduction of new PKS genes into overproducing cells. Numerous modular PKS genes have been cloned and are available for use based on the method of the present invention. The polyketide produced by the PKS enzyme is converted into a hydroxylated polyketide product, an epoxidized polyketide product, a methylated polyketide product, and a glycosylated polyketide product of PKS by a polyketide modifying enzyme, often referred to as a tailoring enzyme. And is further modified. Based on the methods of the present invention, these genes can also be introduced into overproducing host cells to prepare the modified polyketides of interest. The following table lists exemplary PKS genes and lists corresponding enzyme references that may be utilized in accordance with the present invention. Also provided are various references describing polyketide tailoring enzymes and modifying enzymes and corresponding genes that can be used to make the recombinant DNA compounds of the present invention.
[0031]
[Table 1]

Any of the above-mentioned genes, with or without genes for polyketide modification, if any, can be used in the overproducing host cell of the present invention. Furthermore, the host cells of the invention can be constructed by transformation with multiple vectors each containing a portion of the desired PKS and a modified enzyme gene cluster: US Pat. No. 6,697, incorporated herein by reference. See No. 033,883.
[0032]
In a preferred manner, the endogenous PKS gene of the overproducing strain is deleted or otherwise inactivated before or after introduction of the gene encoding the PKS of interest. In one important aspect, the invention relates to genetic overproduction in which the gene encoding PKS that produces the “overproduced” polyketide has been deleted and the gene encoding the PKS of interest can be easily introduced and expressed. A host cell is provided. In certain preferred embodiments, the genetic overproduction host cell is derived from a Saccharopolyspora erythraea host cell that produces erythromycin at a level greater than 2.5 g / L and either a deletion of all or substantially all of the eryA gene or It is modified by any mutational inactivation of the ketosynthase (KS) domain of module 1 of DEBS PKS.
[0033]
To illustrate this aspect of the invention, a genetic block on the polyketide synthase DEBS was introduced into an erythromycin overproducing strain of Saccharopolyspora erythraea that results in non-production of 6-deoxyerythronolide. Expression of the oleAI gene of oleandomycin PKS in this blocked strain was found to produce 15-nor-erythromycin A. The exogenous addition of synthetic molecules to this blocked mutant culture resulted in the production of a novel biologically active C-13 derivative of an erythromycin analog. This latter strategy, referred to as precursor-directed biosynthesis, utilizes biosynthetic blocked PKS mutants that can incorporate chemically directed priming units or intermediates downstream of mutations in PKS. Numerous C-13 derivatives of 6-dEB can be produced in Streptomyces coelicolor by introducing mutations into ketosynthase (KS1) of DEBS module 1 by incorporating a synthetic partial chain into PKS (this book) (See PCT Publication Nos. 99/03986 and 97/02358, which are incorporated herein by reference). However, this methodology is If it can be carried out in an overproducing strain of erythraea, it is advantageous not only in that more polyketides are made, but in that fully glycosylated and hydroxylated compounds are produced.
[0034]
In order to achieve this purpose, plasmid pKOS40-57 was constructed as a delivery vector, and an active site Cys729> Ala mutation was introduced into the KS1 domain of DEBS1 module 1 (see above, PCT publications 99/03986 and 97/02358). In the issue). The inactive KS1 domain prevents the growth of the initiation unit and allows exogenous synthetic diketide thiol esters to be introduced into the PKS (PCT Publication 00, incorporated herein by reference). / 0444717). Saccharopolyspora erythraea K1 protoplasts were transformed with pKOS40-57, and eight transformants were obtained that had no biological activity against Bacillus subtilis. To confirm that pKOS40-57 integration occurred at the appropriate location on the chromosome, genomic DNA was extracted from one of the transformants for PCR analysis. Non-selective growth was then performed that allowed for plasmid eviction via double crossover events. It was found that three-fifths of apramycin-sensitive colonies were in the desired KS1 null (KS1 °) mutant. One such KS1 ° null strain designated K39-14 did not produce any detectable erythromycin or 6-dEB-like product and showed no biological activity when grown in R2YE medium. . This K39-14 strain was used for heterologous expression of the oleAI gene that complements the KS1 null mutant and for precursor-directed biosynthesis of novel erythromycin analogs.
[0035]
Subunits from heterologous modular PKS can be functionally assembled to make hybrid polyketides. For example, both the DEBS3 subunit and the OleA3 subunit (subunit 3 of 8,8a-deoxyoleandolide PKS) can fully complement the picromycin PikAIII and PikAIV subunits (incorporated herein by reference). PCT Publication No. 99/61599). The loading domain of wild-type DEBS from Saccharopolyspora erythraea has been shown to accept acetate to form low levels of 15-nor-erythromycin c. Similar results were observed when DEBS was expressed in Streptomyces coelicolor to form 15-nor-6dEB (1: 5 6dEB), which is incorporated herein by reference. No. 5,672,491). Ole PKS produces 8,8a-deoxyoleadlide, which differs from 6-dEB, which is the product of DEBS with only one carbon in the priming unit. The subunits of Ole PKS and DEBS contain significant structural differences: they are only 45% identical between DEBS and Ole PKS, albeit functionally similar.
[0036]
Expression plasmid pKOS39-110 (this is ermE ^* Expression of the oleAI gene under the control of the promoter is available (see PCT publication 99/061599, incorporated herein by reference) and using the ΦC31 binding site Thus, it was convenient to introduce the oleAI gene into strain K39-14 by integration into the chromosome. Transformation of the Saccharopolyspora erythraea K39-14 plasmid with pKOS39-110 occurred within the strain, with the by-product 15-nor-erythromycin C, 15-nor-erythromycin A about 5-10 mg / L. It showed antibacterial activity. Mass spectra and LC retention times were consistent with known standards. The protonated molecular ionic species at m / z 706 is consistent with the proposed structure of 15-nor-erythromycin C. This result shows that OleAI can complement DEBS1 to some extent. S. The ability to express this heterologous PKS subunit in erythraea also indicates that a heterologous PKS enzyme can be produced in the global overproduction host cell of the present invention.
[0037]
Precursor-directed biosynthesis involves introducing mutations into the DEBS module 1 ketosynthase (KS1) and adding a chemically synthesized N-acetylcysteamine (NAC) thioester of the diketide precursor to the fermentation. In Streptomyces coelicolor to make many C-13 derivatives of 6-dEB. To simplify the production of erythromycin analogs by precursor-directed biosynthesis and to fully process the production of polyketides to erythromycin A analogs, in the chromosome of a Saccharopolyspora erythraea overproducing strain producing strain K39-14 A KS1 ° mutation was introduced into the cell, which was fermented in F1 medium in the presence of 0.25 g / L propyl NAC diketide (4) or vinyl NAC diketide (5) as shown in FIG. (See Brunker et al., 1998, Microbiol. 144: 2441-2448, incorporated by reference). Each of these strains produced the corresponding 15-methylerythromycin or 14,15-dehydroerythromycin derivatives (compounds 6-8 and 9-11 in FIG. 2) between 10-20 mg / L. Both erythromycin analogues showed antibacterial activity against Bacillus subtilis. The structure of 15-methylerythromycin A was determined by NMR.
[0038]
The amount of erythromycin is much lower than the potential of this strain, presumably due to membrane transport barriers, but these yields are sufficient for biological screening from small culture volumes. Given possible sequences of synthetic diketides that can be chemically synthesized, it is possible to produce and screen more erythromycin derivatives in a convenient manner.
[0039]
Based on the method of the present invention, an overproducing strain of Saccharopolyspora erythraea is used to produce various erythromycin compounds that are not naturally produced as the main product by the strain. S. The OleAI complement of the DEBS1 mutant containing the KS1 null mutation in the erythraea overproducing strain is a functional heterologous PKS consisting of proteins derived from two different PKS enzyme complexes, and is mainly produced by some unmodified overproducing strains An example of a PKS that produces a polyketide that is not made as a product is provided. S. Production of 10,11-anhydroerythromycin in an erythraea overproducing strain is a functional heterologous PKS complex comprising a protein comprising a module consisting of enzyme domains derived from two different PKS enzymes and unmodified overproduction Examples of PKS complexes that produce polyketides that are not made by some strains are provided. S. cerevisiae containing a KS1 null mutation Production of polyketides by diketide supplementation in erythraea overproducing strains is a non-functional heterologous PKS complex that can be used to produce polyketides that differ from those produced by wild-type PKS by providing a synthetic chemical starter for PKS Provide examples of the body.
[0040]
Thus, the present invention provides useful methods and host cells for producing many different polyketides in recombinant host cells. However, it is known that polyketide producing host cells that have been optimized through mutagenesis to produce high titers of polyketides are difficult to transform. Furthermore, the low efficiency of transformation observed in such cells decreases significantly as the size of the vector used to transform the cells increases. For very large vectors, transformation is often not simply possible. Since the PKS gene is usually very large (> 30 kb), this low transformation efficiency is a significant barrier to realizing the advantages of the present invention. There is some evidence and speculation that this transformation barrier may be due to a restriction-modification system in the host.
[0041]
Conjugation-mediated transformation may overcome this barrier to some extent; this may be the result of lower sensitivity of single-stranded DNA that occurs upon conjugation to a restriction-modification system. However, even conjugation-mediated transformation cannot succeed with very large vectors that are integrated into the host cell chromosome by homologous recombination. This barrier may be a result of the nature of the homologous recombination event and may be low compared to the cleavage of DNA delivered by the restriction-modification system.
[0042]
Vectors containing binding sites (attP) from phage (eg, phiC31, pSAM, and pSLP vector based vectors) can be integrated into the host cell chromosome by a mechanism mediated by the phage integrase enzyme. However, when this type of vector was used for conjugation-mediated transformation in Saccharopolyspora erythraea host cells, very low transformation efficiencies were observed as described above. The conjugate is analyzed by DNA sequence analysis and the vector is utilized by phages in other organisms and is not present at sites that differ in sequence from the previously identified preferred binding site sequence (attB sequence). I found that it was incorporated into the work. This low transformation efficiency barrier was overcome by altering the host cell chromosome to include the attB sequence. The resulting recombinant host cell can be transformed with relatively high transformation efficiency by conjugation with a vector containing the phiC31 binding site sequence, even with very large vectors containing the entire PKS gene cluster.
[0043]
Accordingly, in certain embodiments, the present invention provides E.I. A recombinant host cell other than a Coli or human host cell is provided, the recombinant cell being modified to contain one or more attB sites compared to the cell from which the cell is derived (herein). See Thorpe et al., 1998, Proc. Natl. Acad. Sci. USA 95: 5505-5510, and Groth et al., 2000, Proc. Natl. Acad. Sci. USA 97: 5995-6000, incorporated by reference in the literature. about). Cells modified to include multiple binding sites may be preferred for the purpose of introducing multiple genes at different locations on the chromosome. In one embodiment, the cells are derived from cells that have been optimized by mutagenesis and produce polyketides at high levels. In another embodiment, the cell is a Saccharopolyspora erythraea host cell such as NRRL2338 or a cell that produces erythromycin in excess of 2.5 g / L.
[0044]
In another embodiment, the present invention provides a method for obtaining a transformant of a cell modified to contain a phiC31 binding site, said method comprising a complementary binding site (attB) with said cell. And a cell containing a vector containing attP are complementary to each other, which can be a phiC31 integrase, so that the gene can be in the vector or in the host cell and under the control of a heterologous promoter. A step of joining in the presence). On the other hand, attP is typically used on the vector and attB on the chromosome, but one can also place attP in the chromosome and attB on the vector according to the method of the invention. In another embodiment, the present invention provides a cell produced by such a method. In a preferred embodiment, cells produced by such methods produce polyketides that are not produced by the cells prior to the aforementioned conjugation step.
[0045]
The following examples are given for the purpose of illustrating the invention and should not be construed as limiting the invention or the claims.
[0046]
(Example 1)
(Strain, medium conditions, and DNA manipulation)
DNA manipulation was performed in Escherichia coli XL1-Blue (Stratagene). The Saccharopolyspora erythraea overproducing strain produced erythromycin in excess of 7 g / L (strain K1). Transformation of this strain was performed according to the standard Streptomyces protoplast method, except that TSB medium was used instead of YEME. Transformants were sorted with thiostrepton (50 μg / mL) or apramycin (100 μg / mL) on R2YE regeneration plates or R5 regeneration plates.
[0047]
(Example 2)
(Construction of Saccharopolyspora erythraea KS1 null mutant)
Plasmid pKOS40-57 was transformed into a DEBS KS1 null mutant (special replacement of alanine with the active site cysteine of the DEBS KS1 domain; Kao et al., 1996, Biochemistry 35: 12363-12368, incorporated herein by reference. The 4.0 kb NdeI-EcoRV fragment containing (see reference) was constructed by introducing it into a suicide vector (pUC18 derivative containing an apramycin resistance conferring gene) as a delivery vector. Plasmid pKOS40-57 was transformed into Saccharopolyspora erythraea K1. The apramycin resistant strain (K1-1) was grown in TSB medium containing apramycin (100 μg / ml) to confirm resistance and integration was confirmed by PCR analysis of chromosomal DNA. For double crossover determination, transformants were passaged twice in the TSB medium without apramycin. Protoplasts were regenerated on R2YE plates. Individual colonies arising on the regeneration plates were then screened for sensitivity to apramycin. Colonies containing the desired mutation have been demonstrated not to produce erythromycin. It was named erythraea K39-14. As described above, when transformed with plasmid pKOS39-110 encoding the oleAI gene, strain K39-14 produces 15-norerythromycin and given propyl NAC diketide (compound 4 in FIG. 3) 15 -Methyl-erythromycin was produced.
[0048]
(Example 3)
(Production and analysis of erythromycin derivatives)
The Saccharopolyspora erythraea strain described herein was grown for 5 days at 30 ° C. in 25 mL of R2YE. Cultures were analyzed by HPLC / MS for erythromycin derivatives. The structure determination was mainly based on the agreement between the predicted structure, mass spectrum and HPLC profile for the engineered strain. For mass spectra and HPLC, see S.M. The erythraea wild type strain was used as a reference. In addition, as described in Example 4, further structure confirmation was performed by the NMR spectrum of 15-methylerythromycin A. Biological activity was assessed by an agar plate diffusion assay using Bacillus subtilis as the indicator bacterium and incubated overnight at 37 ° C. to visualize the area of inhibition.
[0049]
(Example 4)
(Characterization of 15-methylerythromycin A)
Saccharopolyspora erythraea strain K39-114 was fermented in the presence of 0.25 g / L propyl NAC diketide (compound 4 in FIG. 3) given to the culture 24 hours after the start of fermentation as described in Example 3. I let you. 15-Methylerythromycin A extracted from the fermentation was further purified by column chromatography using toluene / acetone 1: 1 for characterization to give a white solid. ¹ H and ¹³ C spectrum is CDCl at 400 MHz (Bruker DRX) ₃ Recorded in solution. Thin layer chromatography was performed on glass pre-coated with silica gel 60 and spots were visualized by vanillin staining and then heated. NMR value: ¹ 1 H NMR (400 MHz, CDCl ₃ ) Δ 5.14 (dd, 1H, J = 2.0, 8.8), 4.91 (d, 1H, J = 4.8), 4.44 (d, 1H, J = 7.2) , 4.05-3.98 (m, 2H), 3.84 (brs, 1H), 3.60 (d, 1H, J = 7.6), 3.54-3.47 (m, 1H) ), 3.34 (s, 3H), 3.25 (dd, 1H, J = 7.2, 10.4,), 3.12 (br q, 1H, J = 7.0), 3.04 (D, 1H, J = 9.6), 2.90 (dq, 1H, J = 7.2, 9.1), 2.77-2.67 (m, 1H), 2.52 (ddd, 1H, J = 4.4, 9.6, 12.4), 2.43-2.37 (m, 1H), 2.34 (s, 6H), 2.05-1.90 (m, 2H) ), 1.90-1.80 (m, 2H), 1.79-1.55 (m, 3H), .50 (s, 3H), 1.36-1.09 (m, 27H), 0.94 (t, 3H, J = 7.2). ¹³ C NMR (100 MHz, CDCl ₃ ) Δ 221.2, 174.6, 102.2, 95.4, 82.5, 79.0, 77.0, 76.3, 76.0, 74.1, 73.5, 71.6, 69.9, 68.0, 64.6, 64.5, 48.5, 44.2, 43.9, 39.2, 38.5, 37.6, 36.8, 34.0, 29. 2, 27.8, 26.1, 20.5, 20.4, 18.5, 17.7, 17.3, 15.2, 14.9, 13.0, 11.0, 8.1.
[0050]
(Example 5)
(Comprehensive production host lacking the native PKS gene)
Many mutations that improve the yield of Saccharopolyspora erythraea high producers are present in chromosomal regions other than the PKS gene, affecting precursor pools, regulatory processes, nutrient utilization, and other factors. Such yield-enhancing mutations can be developed directly and rapidly by using the strain as a global host for the overproduction of mutant and polyketide encoded by heterologous PKS genes. This mainly requires the ability to supply all starting materials used by PKS and coenzymes to make specific polyketides. Fortunately, all modular PKS use one or more small groups of simple carboxylic acids (eg acetate, propionate or butyrate and their 2-carboxy derivatives) to form a carbon skeleton and dideoxyhexide or trideoxyhexose (primary (Made from 1-thymidine diphosphoglucose) to form a glycoside of a simple polyketide. All other tailoring reactions contain common cofactors that are available in most bacterial cells. Therefore, S. erythraea can provide all or most of the starting material; some of the specialized starting materials required for a particular polyketide are introduced by genetic engineering.
[0051]
The DEBS gene from a Saccharopolyspora erythraea erythromycin overproducer can be exchanged with a heterologous PKS from a non-overproducing strain. Enhanced production of metabolites created by the introduced PKS gene can demonstrate that the host actually contains factors for metabolite overproduction that can be developed for the production of other polyketides. Heterologous genes The native DEBS gene can first be deleted prior to introduction into the erythraea overproducing strain. This was accomplished by a two-step homologous recombination procedure, as described in more detail below. The sequence flanking the DEBS gene (approximately 1.5 kb) was amplified by PCR and cloned into a suicide vector with a selectable marker (for apramycin resistance). This strain was transformed with a single crossover selected by using a plasmid and a selectable marker. Colonies were grown without selection and screened for loss of apramycin resistance indicating double crossover recombination. Double-crossing candidate genotypes were confirmed by analysis using PCR, loss of erythromycin production, and Southern blot hybridization. One of the strains thus confirmed was named K97-71.
[0052]
S. erythraea K97-71 contains chromosomal deletions of three eryA genes and insertion of the xylE gene from Pseudomonas aeruginosa in place in the chromosome. To make this strain, plasmid pKOS97-49b was first constructed as follows. Two fragments flanking the eryA gene are S. cerevisiae. PCR amplification from erythraea genomic DNA using primers as follows (SphI, HindIII, BamHI and EcoRI restriction sites are underlined):
eryAI left flank forward direction:
5'-TTT GCATGC GGCCACGCGCCACGTCGTGAACC,
eryAI left flank reverse direction:
5'-TT AAGCTT CATATTCCCCCTACTCGACACCAC;
eryAIII right flank forward direction:
5'-TTT GGATCC GGCGGAGGGAATACATGACCACGAC,
eryAIII right flank reverse direction:
5'-TTTTGAATTCCCGCTCGCTGAAGTCCAGGCT.
The two fragments were then cloned into pSET152 using the underlined restriction sites and corresponding sites in pSET152 (Bierman et al., 1992, Plasmid cloning vectors, incorporated herein by reference. for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp., Gene 116: 43-49). The resulting plasmid was named pKOS97-49a.
The xylE gene was PCR amplified and cloned into the NdeI and XbaI sites of pKOS-97-49a to create pKOS97-49b. This plasmid no longer contains the phiC31 integrase gene and the attP locus from pSET152 and serves as a suicide vector for delivery by homologous recombination.
[0053]
Overproducing strain S. The protoplast of erythraea K1 was generated by standard procedures except that cells were grown in TSB medium and harvested after 3 days of growth (Hopwood et al., 1985, Genetic, incorporated herein by reference). Manifulation of Streptomyces: See A Laboratory Manual. The John Inns Foundation, Norwich, UK). Plasmid pKOS97-49b DNA was obtained from E. coli. Prepared from E. coli ET12567, unmethylated DNA was generated and used for protoplast transformation using standard procedures except using PEG3350 (Sigma) (see Hopwood et al., supra). Plates were covered with 1 mg apramycin and selected for plasmid integration by homologous recombination. Apramycin resistant colonies were analyzed by Southern hybridization to confirm a single crossing at the expected location. One colony was selected to perform the double crossing shown below. The strain was IT agar (5 g glucose per liter, 5 g tryptone, 0.5 g betaine hydrochloride, 5 g corn starch, 1 g corn step liquor (50%), 200 mg MgSO ₄ ・ H ₂ O, 2 mg ZnSO ₄ _7H ₂ O, 0.8 mg CuSO ₄ _5H ₂ O, 0.2 mg CoCl ₂ _6H ₂ O, 4 mg FeSO ₄ _7H ₂ O, 80 mg CaCl ₂ _6H ₂ O, 150mg KH ₂ PO ₄ , 10 g NaCl, 20 g agar) until they were fully spore grown. Spores were collected, diluted on IT plates, and single colonies were screened for apramycin resistance deficiency. One apramycin sensitive colony that did not produce erythromycin was isolated. Double crossing was confirmed by Southern hybridization. This strain was designated as S. It was named erythraea K97-71.
[0054]
Strain K97-71 is a global overproduction host of the present invention that can insert PKS genes for other non-6-dEB polyketides. Because of their large size (30 kb or more) it can be difficult to move a complete set of PKS genes at a time, so its construction can be a series of 2-3 homologous pairs if necessary It can be divided into replacement steps. The picromycin CKS gene serves as an illustration of this aspect of the invention. Picromycin PKS is related to DEBS but produces significantly different compounds. Picromycin PKS synthesizes narbonolide (similar to 6-dEB except that there is no carbon 10 methyl group, a double bond between carbon 10 and 11 and a carbon 3 ketone). These are due to differences in modules 2 and 6 of picromycin PKS. The AT domain of PKS module 2 incorporates acetate instead of propionate, has additional dehydratase (DH) activity in module 2, and no ketoreductase (KR) domain in module 6. Also, the construction of picromycin and DEBS PKS is similar except that module 5 and module 6 of picromycin are divided into two ORFs. The amino acid sequence similarity between the two PKSs is about 40%. At the same time, these characteristic features of picromycin PKS make it an ideal choice to explain that a modular PKS different from DEBS overproduces polyketides when picromycin PKS is transferred to an erythromycin overproducing organism.
[0055]
To introduce a heterologous PKS gene, a plasmid containing an attP binding site can be used. Alternatively, the double recombination procedure described above can be used. Once complete PKS is introduced, the strain is analyzed for production of metabolites. Because the erythromycin deoxysugar biosynthetic gene and glycosyltransferase gene remain intact, depending on the activity of the glycosyltransferase present in the ery gene cluster, the polyketide aglycone (ie, narbonolide), the desosaminylated derivative (narvo) It is possible that either or both of (mycin) may be produced. In addition, either C-6 or C-12 hydroxylation reactions can occur. The total amount of polyketide produced in the parent strain is directly compared to the production level of erythromycin A under similar conditions to indicate overproduction.
[0056]
Other PKS genes can be easily introduced into the strain K97-71 megalomycin PKS gene (PCT patent application number US 00/27433, filed Oct. 4, 2000, incorporated herein by reference). And oleandolide PKS gene (see PCT Publication No. 00/026349, incorporated herein by reference), but is not limited thereto. This host can also be used for the production of polyketides related only to erythromycin, such as FK506 and epothilone. This also ensures the production of a unique substrate for the PKS enzyme (eg 2-ethyl malonate or 2-hydroxymalonate) or modifies the polyketide backbone as it is formed (eg dehydration) (Or oxidation) with the introduction of certain non-PKS genes to provide the enzyme. In addition, various promoters (eg, the act promoter from the erythromycin overproducing strain, ermE ^* Any one of the promoter and eryA promoter) can be used to drive the expression of the heterologous PKS gene cluster.
(Example 6)
(Construction of S. erythraea strain containing PhiC31 binding site)
The Saccharopolyspora erythraea strain does not contain the chromosomal DNA sequence that actually corresponds to the binding site sequence of phiC31. Thus, the efficiency of binding to plasmids derived from phiC31 is not high with such strains, and introduction of very large plasmids into such strains can be difficult if not impossible. As a result, the present invention provides various polyketide producing strains (including but not limited to S. erythraea strains) that have been improved by inserting phiC31 binding site sequences into the chromosomal DNA of the polyketide producing strain.
[0057]
S. erythraea K24-1 is a chromosomal deletion of three eryA genes as well as the attB locus for Streptomyces phage phiC31 from Streptomyces lividans, then ermE ^* Includes insertion in place of the promoter. To create this strain, plasmid pKOS134-04 was first constructed as follows. The phiC31 attB site was inserted between the Hind III and BamH I sites of pKOS97-49a using the following two annealed oligonucleotides:
Forward direction:
5′-AGCTTCGGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGGCGGTAA-CTAGTG,
and
Reverse direction:
5′-GATCCCACTTAGTACCGCGCCGGGGAGCCCAAGGGCACGCCCTGG-CACCCCGA.
This plasmid was named pKOS024-87. Plasmid pKOS0134-04 is the ermE between the resulting SpeI and BamHI sites of pKOS024-87. ^* It was generated by inserting an approximately 300 bp NheI / BamHI fragment containing the promoter.
[0058]
Plasmid pKOS0134-04 was constructed from E. coli as described below. Via a bond from Coli. erythraea K97-71. Screening for single and double crossings by homologous recombination was performed as described above. A double crossover strain confirmed by Southern hybridization was obtained, and S. cerevisiae was obtained. It was named erythraea K24-1.
S. erythraea strains K97-71 and K24-1 are prepared by collecting spores from strains grown on 1-2 IT plates, filtering the spores with sterile cotton and resuspending with 1 ml of 20% glycerol. It became cloudy. The spore suspension was stored at about 20 ° C. A 20 μL aliquot of the spore suspension was added to 5 ml of 2 × YT and incubated at 30 ° C. with shaking. After 1 hour, spores were collected by centrifugation (recipient cells). Donor cells, E. coli / PUZ8002 was prepared by transforming with pSET152 or pKOS97-113 or pKOS97-69a and selecting only apramycin resistance. Plasmids pKOS97-113 and pKOS97-69a are derivatives of pSET152 containing an approximately 30 kb insert carrying the megAI-III and eryAI-III genes, respectively.
[0059]
Several colonies were picked from the primary transformation plate and used to inoculate 5 ml of LB containing chloramphenicol (10 μg / mL), kanamycin (100 μg / mL), apramycin (60 μg / mL). Cells were grown at 37 ° C. for 3-4 hours (0.4-0.6 OD600), collected by centrifugation, washed with 5 ml LB, centrifuged, and resuspended in 100 μL LB. Conjugate transfer between donor cells and recipient cells was performed by resuspending recipient cells in 100 μL of donor suspension and incubating at 37 ° C. for 15-30 minutes without shaking. Cells were plated on R5 plates containing 50 μg / mL nalidixic acid and incubated at 34 ° C. for 16 hours. The plates were then covered with 3 ml soft nutrient agar containing 1 mg nalidixic acid and 2 mg apramycin. Conjugated bodies were observed after an additional 48 hours of incubation.
[0060]
The joining frequency is approximately 5 × 10-5 for K24-1 :: pSET152, 3 × 10-7 for K97-71 :: pSET152, 1 × 10-6 for K24-1 :: pKOS97-113, and K97-71. :: pKOS97-114, which was less than 1 × 10−7.
[0061]
Plasmid integration of the K24-1 :: pSET152 conjugation body and the K97-71 :: pSET152 conjugation body into the chromosome was confirmed as follows. Genomic DNA was prepared from several conjugation completes of each strain, digested with BamHI or EcoRI, religated, and E. coli. Used to transform E. coli. Apramycin was used to select the recircularized DNA containing the pSET152 vector integrated into the chromosome and the attB / attP junction sequence. Plasmid DNA was prepared from apramycin resistant colonies and the attB / attP junction was sequenced using primers annealed upstream of the attP portion in pSET152. In the four separate K24-1 :: pSET152 conjugants analyzed, pSET152 is integrated into four different chromosomal locations, which can be found in other Streptomyces spp. It did not show any similarity with the known attB site. In the seven analyzed K24-1 :: pSET152 conjugates analyzed, pSET152 was incorporated into the engineered phiC31 attB in each case.
[0062]
These results indicate that integration of the vector mediated by integrase phiC31 results in S. cerevisiae without an engineered attB site. erythraea spp. Are not site-specific and occur relatively infrequently. The placement of the appropriate attB or attP sites in the chromosome, as in the example shown in this example, significantly increases the frequency of conjugation of phiC31-based vectors, resulting in site-specific integration.
[0063]
While the invention has been described with the description and examples described, those skilled in the art will appreciate that the invention can be practiced in various embodiments, and the foregoing description and examples are for purposes of illustration, It is understood that the claims are not limited.
[Brief description of the drawings]
[Figure 1]
FIG. 1 shows the modular organization of the three proteins that make up DEBS (DEBS1, DEBS2, and DEBS3), intermediates formed at each step in the synthesis of 6-deoxyerythronolide B (6-dEB), and 6 -Shows important steps in erythromycin biosynthesis, including the structure of erythromycin resulting from correction of dEB (erythromycin C not shown).
[Figure 2]
FIG. 2 shows the replacement of the KR domain of module 2 with the KR and DH domains of module 4 of rapamycin PKS in DEBS and the action of the resulting recombinant PKS and polyketide modifying enzyme in Saccharopolyspora erythraea , Indicate.
[Fig. 3]
FIG. 3 shows the production of erythromycin derivatives by direct biosynthesis from precursors and formed them by providing them to the Saccharopolyspora erythraea host cells of the invention containing two diketides (compounds 4 and 5) and a KS1 ° mutation. The product (compounds 6-11) is shown.

Claims (20)

A method for producing a first polyketide, wherein the method expresses a PKS gene encoding a polyketide synthase (PKS) producing the first polyketide in a cell optimized for the production of a second polyketide A method comprising the steps of: The first polyketide is a derivative of the second polyketide, and the method further comprises modifying a PKS gene in an overproducing cell that is a gene expressing PKS that produces the first polyketide. The method of claim 1. 2. The method of claim 1, wherein the method further comprises the step of introducing a gene expressing PKS that produces the first polyketide in an overproducing cell. The gene expressing PKS producing said second polyketide is deleted or otherwise inactivated before or after introduction of the gene encoding PKS producing said first polyketide. The method described. 2. The method of claim 1, wherein the overproducing cells produce the second polyketide at a level greater than 1 g / L. The method of claim 1, wherein the overproducing cells produce the second polyketide at a level greater than 10 g / L. 2. The method of claim 1, wherein the overproducing cells produce the first polyketide at a level greater than 10 mg / L. 2. The method of claim 1, wherein the overproducing cells produce the first polyketide at a level greater than 100 mg / L. An overproducing host cell useful in the method of claim 1, wherein the gene encoding the second polyketide is deleted and the gene encoding PKS producing the first polyketide is easily introduced. And an overproducing host cell that can be expressed. 10. A host cell according to claim 9, wherein the host cell is derived from a Saccharopolyspora erythraea host cell producing erythromycin at a level above 2.5 g / L and is modified by all or substantially all deletions of the eryA gene , Host cells. 10. A host cell according to claim 9, which is derived from a Saccharopolyspora erythraea host cell producing erythromycin at a level of more than 2.5 g / L, and is mutagenic inactive in the ketosynthase (KS) domain of module 1 of DEBS PKS. A host cell that is modified by conversion. 10. The host cell according to claim 9, which produces 10,11-anhydro-6-deoxyerythronolide B. A recombinant host cell that has been modified to contain one or more attB or attP sites in relation to cells from the derived host cell. 14. The cell of claim 13, wherein the cell is derived from a cell optimized by mutagenesis to produce polyketides at a high level. 14. The cell of claim 13, which is a Saccharopolyspora erythraea host cell. 16. The cell of claim 15, which is derived from Saccharopolyspora erythraea NRRL2338. 16. The cell of claim 15, wherein the cell is derived from a cell that produces erythromycin in excess of 2.5 g / L. 14. A method for obtaining the conversion of a cell according to claim 13, comprising the step of combining said cell with a cell comprising a vector constituting a complementary binding site in the presence of an integrase. A cell produced by the method of claim 18. 20. The cell of claim 19, wherein the cell produces a polyketide that is not produced by the cell prior to the binding step.

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